(219) Excitatory to Inhibitory Transition in GABAergic Currents Guides Circuit Formation of Cortical Interneurons

This abstract is a recipient of the Young Investigator Award
This abstract has been invited to present during the Investigators Workshop Platform poster session

Rationale: Excitatory GABA is a crucial developmental cue that guides perinatal neuronal migration, synaptogenesis, and circuit formation. The switch from embryonic depolarizing GABA to mature hyperpolarizing responses is largely determined by the onset of KCC2 expression. Prematurely upregulated KCC2 function produces marked abnormalities in brain development, especially in GABAergic cortical interneurons (INs). Conversely, we investigated how indefinitely-prolonged depolarizing GABA responses in INs adversely affect cortical circuit development using an IN-specific KCC2 knockout (KO).

Methods: We created the KO by crossing the IN-specific Dlx5:cre-IRES-eGFP and the KCC2^flox lines. We measured seizure susceptibility by latency to onset of flurothyl-induced GTC seizure. For evaluating INs and synapses, we focused on P12-14 barrel field. We recorded sIPSCs using whole-cell voltage clamp in layer 5 (L5) pyramidal neurons in acute brain slices. We used immunohistochemistry and epifluorescence/confocal imaging to visualize expression of KCC2 and GABA_A receptor subunits α1-5, and IN distribution by Dlx5-driven reporters and somatostatin (SST)/parvalbumin (PV) antibodies.

Results: KO mice exhibit abnormal neurological development, including: spontaneous seizures, a 45% faster onset of flurothyl-induced seizures, reduced body weight, and late postnatal mortality, but normal gross and histological anatomy of organs, including feeding structures and the brain. We observed the earliest cortical KCC2 expression in L5 INs, as early as E16-18 in wild-type mice. We expected these cells to be the most affected by loss of KCC2, and indeed found a 30% increase in density of INs specific to L5. However, we observed that the INs and inhibitory synapses of KO animals appear normal in many aspects: normal distribution of immature Dlx5+ INs, normal frequency of L5 principal cell sIPSCs, and GABA_A receptor subunit expression across the cortical layers.We postulated that the effects of KCC2 loss might be specific to IN subtypes, which in L5 consist mainly of SST and PV INs with distinctly different circuit functions and developmental sequences. We found opposing changes in densities of these subtypes: a 12% increase in L5 density of SST INs in KO, but a decrease in PV IN density in L2-4 and L6. Within L5, PV IN density was unaffected, corroborating normal pyramidal cell sIPSCs, which reflect somatic and proximal dendrite input of predominantly PV IN projections.

Conclusions: Cortical INs are vital in regulating neuronal excitability and circuit function, and improper GABAergic excitation of INs underlies genetic and acquired epilepsies. Our data show that loss of KCC2 in developing INs causes failure to thrive and promotes seizures, which may be a result of altered IN distribution. To better understand the developmental origins of this abnormal distribution, we are now investigating (1) the early distribution of INs in the KO that precedes the changes in SST/PV distribution, and (2) whether GABAergic responses are anachronously excitatory in maturing INs in KO.

Funding: Please list any funding that was received in support of this abstract.: This research is supported by VA merit award 1I01BX001189.